The present invention relates a novel DNA derived from a gene encoding rice adenylate kinase which has a promoter function in the plant body or plant cell and expression of a foreign gene and its regulation using the DNA.
It is generally known that regulation of a structural gene is associated with a region called a promoter which is located upstream of the structural gene. A promoter is a DNA sequence which is located upstream of a structural gene and contains a signal (the TATA box) which directs an RNA polymerase to initiate transcription toward subsequent protein synthesis.
A specific nucleotide sequence called a cis element which is located upstream of the TATA box is supposed to play an important role in regulation of the strength and expression of the promoter.
For example, deletion studies and DNA-fusion studies on the promoter of a drought-inducible gene (rd29A) isolated from Arabidopsis thaliana [Koizumi et al., Gene 129:175-182(1993)] revealed that the cis element that regulates the drought-inducible gene rd29A is a 9-bp sequence of TACCGACAT [Yamaguchi-Shiozaki et al., J. Plant Res., 108:127-136(1995)], and that the above-mentioned 9-bp cis element is essential to the drought-inducibility of the promoter.
On the other hand, the 35S promoter from cauliflower mosaic virus [Guilley et al., Cell 30:763-773(1982)] is one of the well-known promoters for plant transformation and regulates expression of foreign genes introduced into dicotyledonous plants and protoplasts [From et al., Proc. Natl. Acad. Sci., 82:5824-5828(1985)]. Analysis in tobacco [Morell et al., Nature 315:200-204(1985)] and petunia [Sander, Nucl. Acid Res. 15:1543-1558(1987)] demonstrated that the 35S promoter is more than 30 times as strong a promoter as the nopaline synthase promoter. Because of its potent promoter activity in dicotyledons, the 35S promoter is often used for efficient expression of plant-viable foreign structural genes introduced into dicotyledons.
However, the 35S promoter only shows relatively weak activity in agriculturally important gramineous monocotyledons [Hauptmann et al., Plant Cell Rep., 6:265-270(1987)].
In contrast, the promoter of the maize alcohol dehydrogenase (Adh) gene allows a very low level of expression in protoplasts of dicotyledonous Nicotiana plumbaginifolia [Ellis et al., EMBO J., 6:11-16(1987)].
Expression of the 35S promoter and the Adh promoter does not seem amenable to tissue-specific or chemical regulation.
Techniques for efficient expression of foreign structural genes and for regulation of their expression are important to create new practically useful varieties of a wide range of plants by gene recombination in the future.
Hence, there is a demand for a promoter that shows promoter activity in monocotyledons as well as in dicotyledons and is easy to regulate tissue- or site-specifically or by harmless chemicals.
On the other hand, it is known that adenylate kinase (AK) is involved in the energy system in animals, microorganisms and plants [Noda et al., The Enzymes., Academic Press, New York:279-305(1973)].
With respect to plants, induction of adenylate kinase by sucrose in rice [Kawai et al., Ikushugakuzasshi, suppl. 1:253(1994)] and localization of the enzyme in vascular bundles [Kawai et al., J. Plant Physiol. 146:239-242(1995); Kawai et al., Plant Molecular Biology 27:943-951(1995)] have been disclosed.
Two cDNA species encoding rice AK (AK-a and AK-b) have been isolated [Kawai et al., The Plant Journal 2(6):845-854(1992)]. However, the genomic DNA corresponding to them have not been obtained, and the nucleotide sequence of their promoter region remains unknown.
The object of the present invention is to obtain and provide a novel DNA fragment having a promoter function which enables expression of a fused structural gene in a plant body or plant cells and regulation of the expression by a harmless chemical substance or tissue- or site-specifically.
As a result of their extensive research to solve the above-mentioned problems, the present inventors isolated a promoter region of the rice AK-a gene and found that the promoter region functions as a promoter in other plants to express a foreign gene and regulate its expression. The present invention was accomplished on the basis of the discovery.
The present invention provides a promoter of a gene encoding rice adenylate kinase described below, or a DNA fragment having a promoter function which consists of at least part of the nucleotide sequence shown in SEQ ID NO:1 in the Sequence Listing wherein one or more bases may be deleted, added or replaced as long as the fragment having a function to regulate expression of a structural gene viable in a plant, for example, a DNA of 1443 bp shown in SEQ ID NO:2. The present invention also provides a vector containing the DNA fragment having a promoter function, a bacterium containing the vector and a plant transformed with the vector or the bacterium. The present invention still further provides a method for expression of regulating a recombinant DNA which comprises fusing the DNA fragment having a promoter function to another DNA sequence and introducing the resulting recombinant DNA into a plant.